In eukaryotes including plants, the progression of cell cycle events is regulated by a network of gene products and factors to ensure that this crucial process is initiated as an integral part of the growth and the developmental program, and in response to the external environment. These factors exert their influences on the cell cycle machinery via various pathways. At the center of the machinery lies an enzyme complex consisting of a catalytic subunit, cyclin-dependent protein kinase (CDK), and a regulatory subunit, cyclin. CDKs are a group of related serine/threonine kinases and their activity generally depends on their association with cyclins (Pines, 1995).
Early work disclosed the existence of CDKs in yeast. A CDK called Cdc2 (p34cdc2, or CDK1) was identified in fission yeast Schizosaccharomyces pombe (Hindley and Phear, 1984) and a Cdc2 homolog called CDC28 was identified in budding yeast Saccharomyces cerevisiae (Lörincz and Reed, 1984). In yeast, Cdc2 (or CDC28) kinase appears to be solely responsible for regulating the progression of the cell cycle.
Animal cells have evolved several Cdc2-related CDKs in order to achieve more complex regulation at multiple levels. In mammalian cells, seven distinct CDKs and eight types of cyclins have been identified (see review by Pines, 1995). Complexes of these CDKs and cyclins appear to act sequentially at different checkpoints during the cell cycle, while incorporating the input of different developmental and environmental cues.
Plants, like higher animals, have multiple CDKs (Francis and Halford, 1995; Jacobs, 1995) and cyclins (Renaudin et al., 1996). In Arabidopsis thaliana, at least two Cdc2 homologues, Cdc2a and Cdc2b (Ferreira et al., 1991; Hirahama et al., 1991) and as many as twelve cyclins belonging to three groups (Renaudin et al., 1996) have so far been documented. Of the two Cdc2 homologues in A. thaliana, Cdc2a resembles more closely Cdc2 homologues from other species because it has a conserved PSTAIRE motif and is able to genetically complement yeast cdc2 or CDC28 mutants (Ferreira et al., 1991; Hirahama et al., 1991), indicating some functional homology of A. thaliana Cdc2a with the yeast Cdc2 kinase. Expression analyses showed that A. thaliana cdc2a expression was correlated with the “competence” of a cell to divide and preceded the re-entry of differentiated cells into the cell division cycle (Martinez et al., 1992; Hemerly et al., 1993), and expression of a dominant negative cdc2a mutant resulted in cell cycle arrest (Hemerly et al., 1995). A. thaliana Cdc2b is atypical in that it has a PPTALRE motif in place of the PSTAIRE motif. Like cdc2a, cdc2b is also expressed in dividing plant cells. While cdc2a is expressed constitutively throughout the cell cycle, cdc2b is reportedly expressed preferably in S and G2 phases (Segers et al., 1996).
Relatively little is known about the cyclins and other proteins and factors which regulate the activity of CDK-cyclin complexes in plant cells. Results from yeast and mammalian studies have demonstrated multiple pathways, both positive and negative, by which CDK activity can be modulated (Lees, 1995). In addition to binding by a cyclin, for example, activation of CDKs may also involve a CDK-activating kinase (CAK) which itself is a CDK, and CDC25 protein phosphatase.
A new aspect of regulating CDK activity was discovered with the identification of CDK inhibitors (see reviews by Pines, 1995; Sherr and Roberts, 1995; Harper and Ellege, 1996). These small molecular weight proteins are understood to bind stoichiometrically to negatively regulate the activity of CDKs. It has been suggested that these inhibitors may be involved in animal development and cancer, in addition to their role in cell cycle regulation (Harper and Elledge, 1996). A plant CDK inhibitor activity was observed and was suggested to be involved in endosperm development in maize (Grafi and Larkins, 1995).
The activity of CDK inhibitors has been studied in animals. Transgenic mice have been generated lacking p21, p27 and p57 CDK inhibitor genes. The p21 knockout mice are reported to develop normally but are deficient in G1 checkpoint control, such as cell cycle arrest in response to DNA damage (Deng et al., 1995). Analysis of p27 knockout mice from three independent studies show that transgenic mice lacking p27 display larger body size than control mice (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). The enhanced growth is reportedly due to an increase in cell number (Kiyokawa et al., 1996) and is gene dose-dependent (Fero et al., 1996). In comparison, none of p21 or p57 knockout display enhanced growth. The transgenic mice lacking p57 show a range of developmental defects such as defective abdominal muscles, cleft palate and renal medullary dysplasia (Yan et al., 1997; Zhang et al., 1997). A few developmental defects were observed in p27-/- mice. They include impaired ovarian follicles (thus female sterility), impaired luteal cell differentiation and a disordered estrus cycle. These results reflect a disturbance of the hypothalamic-pituitary-ovarian axis. In comparison, transgenic mice lacking p21 appear to develop normally at both gross anatomic and histologic levels (Deng et al., 1995). In addition, an increase in apoptosis is observed in mice lacking p57. The CDK inhibitor p27 was over-expressed in mouse hepatocytes (Wu et al., 1996), resulting in a general a decrease in overall number of adult hepatocytes which result in aberrant tissue organization, body growth and mortality.
Despite the general conservation of basic cell cycle machinery in eukaryotes, the role of plant cell division during plant growth and development is characteristically different from other eucaryotic cells. In many respects, the regulation of plant cell division and growth can be regarded as distinct from other eucaryotic cells. For example, plant cells are not mobile during morphogenesis. Different sets of hormones are involved in modulating plant growth and development. Plant cells are remarkable for their ability to re-enter the cell cycle following differentiation. Also, cell division in plants is continuous, along with organ formation, and plant body size (the number of total cells and size of the cells) can vary dramatically under different conditions. Plants also have an inherent ability to incorporate additional growth into normal developmental patterns, as is illustrated by a study showing that ectopic expression of a mitotic cyclin driven by the cdc2a promoter resulted in a larger but normal root system (Doerner et al., 1996). However, relatively little is known about the connections of the regulatory genes controlling cell division patterns to the cell cycle regulators such as the CDKs and the cyclins in plants (Meyerowitz, 1997).
A few studies of transgenic expression of cell cycle genes in plants are documented using various cell cycle genes other than CDK inhibitors. A heterologous yeast cdc25, a mitotic inducer gene, was introduced into tobacco plants under the control of a constitutive CaMV 35s promoter (Bell et al., 1993). Transgenic tobacco plants showed abnormal leaves (lengthened and twisted lamina, pocketed interveinal regions), abnormal flowers, and also precocious flowering. Analysis of cell size in the root meristem revealed that transgenic plants expressing the yeast cdc25 had much smaller cells (Bell et al., 1993). The wild type cdc2a gene and variants of dominant negative mutations under the control of CaMV 35s promoter have been used to transform tobacco and Arabidopsis plants (Hemerly et al., 1995). Constitutive expression of wild-type and mutant Cdc2a did not significantly alter the development of the transgenic plants. For the dominant negative Cdc2a mutant, it was not possible to regenerate Arabidopsis plants. Some tobacco plants expressing this construct were obtained and they had considerably fewer but much larger cells. These cells, however, underwent normal differentiation. Morphogenesis, histogenesis and developmental timing were unaffected (Hemerly et al., 1995). As mentioned above, ectopic expression of an Arabidopsis mitotic cyclin gene, cyc1At, under the control of the cdc2a promoter increases growth without altering the pattern of lateral root development in Arabidopsis plants (Doerner et al., 1996).
The yeast two-hybrid system has been used to identify the cyclin-dependent kinase inhibitor gene ICK1 from a plant (Wang et al., 1997). ICK1 is different in sequence, structure and inhibitory properties from known mammalian CDK inhibitors. It has been shown that recombinant protein produced from this gene in bacteria is able to inhibit plant Cdc2-like kinase activity in vitro (Wang et al., 1997).
Cytotoxin genes, i.e. genes encoding a protein which will cause cell death, have been tested in transgenic plants for genetic ablation of specific cells or cell lines during development, including RNase (Mariani et al., 1990), DTT (diphitheria toxin) chain A (Thorsness et al., 1991; Czako et al., 1992), Exotoxin A (Koning et al., 1992) and ribosomal inhibitor proteins (U.S. Pat. No. 5,723,765 issued Mar. 3, 1998 to Oliver et al.). Several disadvantages may be associated with the use of cytotoxin genes for modification of transgenic plants, particularly plants of agronomic importance. The action of the cytotoxin may not be specific and may result in non-specific destruction of plant cells. This effect may be the result of diffusion of the cytotoxin, or of non-specific expression of the cytotoxin gene in non-target tissues. Non-specific low-level expression of the cytotoxin may be a difficult problem to overcome, since most tissue-specific promoters have some levels of expression in other tissues in addition to a high level of expression in a particular tissue. Expression of a potent cytotoxin gene even at a low concentration may have a negative impact on growth and development in non-target tissues. The presence of cytotoxic proteins of transgenic origin may also have a negative effect on the marketability of an edible plant, or plant product, even if the cytotoxin is demonstrably benign to consumers.